UHPLC-MS/MS Quantification of Human Cytosolic Aldehyde Oxidase: Heat-Assisted In-Solution Protein Denaturation and...

The quality of protein denaturation and digestion is critical in determining the performance of a UHPLC-MS/MS method in absolute protein quantification. Previous UHPLC-MS/MS methods on human aldehyde oxidase (AOX1) protein provided little or no methodological information on protein denaturation and digestion. Therefore, we developed and optimized a sample preparation method for the UHPLC-MS/MS quantification of human cytosolic AOX1.

Various reagents and conditions in protein denaturation (urea, Rapigest, temperature, heating time, and acetonitrile) and digestion (incubation time) were evaluated. A UHPLC-MS/MS method was developed and validated for quantifying the surrogate peptide ⁴⁴⁶VFFGEGDGIIR⁴⁵⁶ for AOX1 and its stable-isotope-labelled internal standard.

Surrogate peptide yield was improved by our optimized conditions for AOX1 protein denaturation (heating at 80°C for 15 min together with reduction by DL-dithiothreitol, acetonitrile not added during trypsin digestion) and protein digestion (4 h). A solvent composition of 40% water-10% acetonitrile-50% ammonium bicarbonate buffer enhanced peptide solubility and assay linearity and sensitivity. An AOX1-negative cytosol was an appropriate matrix blank for cellular samples. The UPHLC-MS/MS assay validation metrics were within acceptable FDA guidelines, highlighted by peptide stability during sample preparation, a shorter run time (11.5 min), greater sensitivity (7.5 fmol), broader linearity range (7.5-10 000 fmol), allowing for the low femtomole quantification of human cytosolic AOX1.

Our novel methodological advances, as highlighted by a high temperature, in-solution protein denaturation protocol and optimal digestion time, solvent composition, and matrix blank, enabled the femtomole quantification of AOX1 protein using a validated UHPLC-MS/MS method with better assay performance than previous methods.